The natural history of cutaneous propionibacteria, and reclassification of selected species within the genus Propionibacterium to the proposed novel genera Acidipropionibacterium gen. nov., Cutibacterium gen. nov. and Pseudopropionibacterium gen. nov.

Taxonomy and identification of fastidious Gram negatives are evolving and challenging. We compared identifications achieved with the Vitek 2 Neisseria-Haemophilus (NH) card and partial 16S rRNA gene sequence (526 bp stretch) analysis with identifications obtained with extensive phenotypic characterization using 100 fastidious Gram negative bacteria. Seventy-five strains represented 21 of the 26 taxa included in the Vitek 2 NH database and 25 strains represented related species not included in the database. Of the 100 strains, 31 were the type strains of the species. Vitek 2 NH identification results: 48 of 75 database strains were correctly identified, 11 strains gave `low discrimination´, seven strains were unidentified, and nine strains were misidentified. Identification of 25 non-database strains resulted in 14 strains incorrectly identified as belonging to species in the database. Partial 16S rRNA gene sequence analysis results: For 76 strains phenotypic and sequencing identifications were identical, for 23 strains the sequencing identifications were either probable or possible, and for one strain only the genus was confirmed. Thus, the Vitek 2 NH system identifies most of the commonly occurring species included in the database. Some strains of rarely occurring species and strains of non-database species closely related to database species cause problems. Partial 16S rRNA gene sequence analysis performs well, but does not always suffice, additional phenotypical characterization being useful for final identification.

A novel actinobacterium, designated strain NEAU-THZ27T, was isolated from soil collected from the Cornel peak in Jiaozuo, Henan Province, PR China and characterized using a polyphasic approach. Morphological and chemotaxonomic characteristics of the strain coincided with those of members of the genusKribbella. The results of 16S rRNA gene sequence analysis showed that strain NEAU-THZ27T belongs to the genus Kribbella and was most closely related to Kribbella podocarpi YPL1T (98.96 %), Kribbella karoonensis Q41T (98.89 %), Kribbella aluminosa HKI 0478T (98.86%) and Kribbella hippodromi S1.4T (98.85 %), similarities to other type strains of species of the genus Kribbella were found to be less than 98.7 %. Phylogenetic analyses using the 16S rRNA gene sequence and multilocus sequence analysis using the concatenated gene sequences of the gyrB, rpoB, recA, relA and atpD genes all showed that the strain formed a separate branch in the genus Kribbella. The cell wall contained ll-diaminopimelic acid as the major diamino acid and the whole-cell hydrolysates were ribose and glucose. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol. The predominant menaquinone was MK-9(H4). Major fatty acids were iso-C16 : 0, iso-C14 : 0 and anteiso-C15 : 0, these chemotaxonomic data supported the affiliation of strain NEAU-THZ27T to the genus Kribbella. The DNA G+C content was 68.0 mol%. Furthermore, the strain could be clearly distinguished by concatenated gene genetic distances, the combination of DNA-DNA hybridization results and some phenotypic characteristics. Therefore, it is proposed that strain NEAU-THZ27T represents a novel species of the genus Kribbella, for which the name Kribbella jiaozuonensis sp. nov. is proposed. The type strain is NEAU-THZ27T (=CGMCC 4.7504T=DSM 105535T).

49 - The Cutaneous Propionibacteria

A strictly anaerobic, Gram-negative, non-spore-forming bacterium, designated GH1(T), was isolated from the rumen of Korean native cattle (HanWoo). Cells were straight to slightly curved rods (2.0-4.5 µm long) and were motile by peritrichous flagella. The isolate grew at 30-45 °C (optimum 40 °C), at pH 5.5-6.5 (optimum pH 6.0) and with up to 3.5% (w/v) NaCl. Strain GH1(T) produced acid from d-glucose, d-ribose and d-xylose, with butyric acid being the major end product. The genomic DNA G+C content was 54.6 mol%. Based on comparative 16S rRNA gene sequence analysis, strain GH1(T) was most closely related to Oscillibacter valericigenes Sjm18-20(T) (97.3% 16S rRNA gene sequence similarity). DNA-DNA hybridization between strain GH1(T) and O. valericigenes DSM 18026(T) showed 24% reassociation. The major fatty acids were iso-C13:0 (13.0%), iso-C15:0 (17.6%), anteiso-C15:0 (8.4%) and C14:0 (4.1%), and the cellular fatty acid methyl esters as dimethylacetals (DMAs) were C16:0 DMA (17.8%), iso-C15:0 DMA (15.2%) and C14:0 DMA (4.52%). The cell-wall peptidoglycan of strain GH1(T) contained meso-diaminopimelic acid and the major cell-wall sugar was galactose. Based on 16S rRNA gene sequence similarity, phylogenetic analysis, DNA G+C content, DNA-DNA relatedness and distinct phenotypic characteristics, strain GH1(T) is classified in the genus Oscillibacter as a member of a novel species, for which the name Oscillibacter ruminantium sp. nov. is proposed. The type strain is GH1(T) (=KCTC 15176(T)=NBRC 108824(T)=JCM 18333(T)).

Phenotypic and phylogenetic analysis was performed on four strains of a previously undescribed Gram-positive, rod-shaped, anaerobic bacterium isolated from the rumen and faeces of cattle. This bacterium fermented glucose primarily to lactic acid along with minor amounts of acetic and butyric acids. The four strains produced a temperature-sensitive bacteriocin-like inhibitory substance. Comparative 16S rRNA gene sequence analysis indicated that the bacterium was a member of the clostridial XIVa cluster of the low-G+C content Gram-positive bacteria. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be assigned to a new genus, Lachnobacterium, as Lachnobacterium bovis gen. nov., sp. nov. The type strain is YZ 87T (= ATCC BAA-151T = DSM 14045T = LRC 5382T). Its G+C content is 33.9 mol %.

Phylogenetic analysis and phenotypic characterization were used to assign a multicellular magnetotactic prokaryote the name 'Candidatus Magnetoglobus multicellularis'. 'Candidatus Magnetoglobus multicellularis' lives in a large hypersaline coastal lagoon from Brazil and has properties that are unique among prokaryotes. It consists of a compact assembly or aggregate of flagellated bacterial cells, highly organized in a sphere, that swim in either helical or straight trajectories. The life cycle of 'Candidatus Magnetoglobus multicellularis' is completely multicellular, in which one aggregate grows by enlarging the size of its cells and approximately doubling the volume of the whole organism. Cells then divide synchronously, maintaining the spherical arrangement; finally the cells separate into two identical aggregates. Phylogenetic 16S rRNA gene sequence analysis showed that 'Candidatus Magnetoglobus multicellularis' is related to the dissimilatory sulfate-reducing bacteria within the Deltaproteobacteria and to other previously described, but not yet well characterized, multicellular magnetotactic prokaryotes.

A novel actinomycete strain, DAS-139T, was isolated from a soil sample collected from Gulbarga, Karnataka Province, India. The isolate was characterized by white to grey aerial mycelium. Long spore chains were found on the aerial mycelium and the aerial mycelium was composed of non-motile spores with hairy surfaces. The cell wall of strain DAS-139T contained ll-diaminopimelic acid isomer as the diagnostic diaminoacid indicating that the cell wall was of chemotype-I. The predominant menaquinones were MK-9(H6) (76 %), MK-9(H4) (14 %) and MK-9(H8) (10 %). Phosphatidylethanolamine was the diagnostic phospholipid. On the basis of 16S rRNA gene sequence, phenotypic and phylogenetic analyses, the novel strain was identified as a member of the genus Streptomyces. The novel strain grew optimally at 28 degrees C and pH 9.0. The G+C content of the genomic DNA was 71.8 mol%. 16S rRNA gene sequence analysis showed that the novel isolate had 99.4 % sequence similarity with Streptomyces scabiei ATCC 49173T and 99.2 % similarity with Streptomyces diastachromogenes ATCC 12309T. Furthermore, DNA-DNA hybridization with these two Streptomyces species showed 36.0 and 43.0 % relatedness, respectively. Based on these observations, strain DAS-139T is proposed to represent a novel species of the genus Streptomyces, for which the name Streptomyces deccanensis sp. nov. is proposed with the type strain DAS-139T (=KCTC 19241T=CCTCC AA 207004T).

Tioman Island is one of many sources for underexplored actinobacterial diversity in Malaysia. Selective isolation, molecular profiling, 16S rRNA gene sequencing and phylogenetic analyses were carried out to highlight the diversity of the marine actinobacterial community in a sediment collected off Tioman Island. A high number of diverse actinobacteria were recovered using skim milk/HEPES pre-treatment on a mannitol-based medium. A total of 123 actinobacterial strains were isolated, including thirty obligate marine actinobacteria putatively identified as Salinispora spp. Molecular fingerprinting profiles obtained with a double digestion approach grouped the remaining non-Salinispora-like strains into 24 different clusters, with Streptomyces and Blastococcus as the major clusters. A total of 17 strains were identified as novel actinobacterial species within the genera Streptomyces (n=6), Blastococcus (n=5), Marinactinospora (n = 3), Nocardiopsis (n=1), Agromyces (n=1) and Nonomuraea (n=1) based on 16S rRNA gene sequence analyses. Polyphasic data from three putative Marinactinospora spp. showed that the strains represent a new genus in the Nocardiopsaceae family. Crude extracts from the strains were also found to inhibit the growth of Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Providencia alcalifaciens) pathogens. Hierarchical clustering of the bioactivities of an active fraction revealed a unique profile, which is closely related that of fosfomycin.

Mageeibacillus indolicus gen. nov., sp. nov.: A novel bacterium isolated from the female genital tract

Lactobacillus kosoi Chiou et al. 2018 and Lactobacillus micheneri McFrederick et al. 2018 are closely related, and they share 100 % 16S rRNA gene sequence similarity, 99.6 % pheS gene sequence similarity, 100 % rpoA gene sequence similarity, 97.3 % average nucleotide identity (ANI) value and 76.6 % in silico DNA-DNA hybridization (isDDH) value, indicating that they represent the same species. Fatty acid methyl esters (FAME) analysis and phenotypic characterization also indicated that L. kosoi and L. micheneri are very similar. We propose L. kosoi Chiou et al. 2018 as a later heterotypic synonym of L. micheneri McFrederick et al. 2018. The taxonomic position of Lactobacillus plantarum subsp. argentoratensis in the L. plantarum group was re-examined using a polyphasic approach, including sequence analyses of 16S rRNA, pheS, rpoA and recA genes, average nucleotide identity analysis, in silico DNA-DNA hybridization, fatty acid methyl ester analysis and phenotypic characterization. Results of 16S rRNA gene sequence analysis indicated that L. plantarum subsp. argentoratensis was closely related to L. plantarum subsp. plantarum, L. pentosus and L. paraplantarum in the L. plantarum group, sharing 99.6-99.7 % 16S rRNA gene sequence similarities. Results of pheS, rpoA and recA gene sequence analyses indicated that L. plantarum subsp. argentoratensis was most closely related to L. plantarum subsp. plantarum, having 91.8 % pheS gene sequence similarity, 98.9 % rpoA gene sequence similarity and 93.1 % recA gene sequence similarity. L. plantarum subsp. argentoratensis DSM 16365T shared 95.6 % ANI value and 62.9 % isDDH value with L. plantarum subsp. plantarum ATCC 14917T. The low isDDH value confirmed that L. plantarum subsp. argentoratensis and L. plantarum subsp. plantarum represent two different species, rather than two different subspecies in the L. plantarum group. On the basis of the data from polyphasic characterization obtained in the present study and in previous studies, L. plantarum subsp. argentoratensis is elevated to the species level and represents a novel species of the genus Lactobacillus, for which the name Lactobacillus argentoratensis sp. nov. is proposed and the type strain is DKO 22T (=CIP 108320T=DSM 16365T=JCM 16169T). Two novel Gram-stain-positive bacterial strains, designated 1206-1T and F027-1-2, were isolated from traditional pickle in Heilongjiang Province, PR China, and from the intestinal tract of a honey bee (Apis mellifera) in Hubei Province, PR China, respectively. The two bacteria were characterized by a polyphasic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, fatty acid methyl ester analysis, average nucleotide identity analysis, in silico DNA-DNA hybridization analysis and an analysis of phenotypic features. The results of 16S rRNA gene sequence analysis indicated that strains 1206-1T and F027-1-2 were distantly related to Lactobacillus sharpeae, Lactobacillus hulanensis, Lactobacillus songhuajiangensis, Lactobacillus pantheris, Lactobacillus thailandensis, Lactobacillus camelliae, Lactobacillus jixianensis, Lactobacillus nasuensis, Lactobacillus baoqingensis, Lactobacillus manihotivorans and Lactobacillus porcinae. Strain 1206-1T exhibited 94.2-96.4 % 16S rRNA gene sequence similarities, 69.5-83.3 % pheS gene sequence similarities and 73.1-90.3 % rpoA gene sequence similarities to type strains of phylogenetically related species. ANI and isDDH values between strain 1206-1T and the type strains of phylogenetically related species were 52.7-73.7 % and 21.1-30.1 %, respectively. On the basis of the data obtained in the present study, a novel species, Lactobacillus zhaodongensis sp. nov. is proposed and the type strain is 1206-1T (=CCM 8981T=CCTCC AB 2019200T=LMG 31620T).

Twenty-six strains of acetic acid bacteria were isolated from fruits, flowers and related materials collected in Thailand. They were divided into three genera, Acetobacter, Gluconobacter and Asaia, by phenotypic characterization and 16S rRNA gene sequence analyses. On the basis of 16S-23S rRNA gene internal transcribed spacer (16S-23S rDNA ITS) restriction and 16S rRNA gene sequence analyses, fourteen isolates assigned to the genus Acetobacter were divided into five groups: 1) Group 1A or A. tropicalis (one isolate); 2) Group 2A or A. orientalis (four isolates); 3) Group 3A or A. pasteurianus (five isolates); 4) Group 4A or A. syzygii (one isolate); and 5) Group 5A or A. ghanensis (three isolates). The eleven isolates assigned to the genus Gluconobacter were divided into three groups: 6) Group 1B or G. frateurii (four isolates); 7) Group 2B or G. japonicus (six isolates); and 8) Group 3B or unidentified (one isolate). The remaining isolate was placed into: 9) Group 1C or unidentified, which was assigned to the genus Asaia and considered to constitute a new species on the basis of the 16S rRNA gene sequence analysis and DNA-DNA hybridization.

No common, unique genetic markers applicable to classification and phylogenetics for significant genera within the Propionibacteriaceae family have been suggested yet. Therefore, the aim of the study was to propose those genes in the genera Acidipropionibacterium, Cutibacterium, Propionibacterium and Pseudopropionibacterium. These genera were recently elicited from the genus Propionibacterium through whole genomic analyses. Three housekeeping genes, glyS, infB and rplB, were selected from many others according to the requirements for appropriate classification/phylogenetic markers. Concrete fragments of the genes were amplified using specific primers in most of the type (14) and 11 wild strains (originating from dairy products, human skin and the crop of a laying hen) recently classified into the genus Propionibacterium. Sequences obtained from amplicons were used to perform gene statistics and phylogenetic analyses with respect to applicability in classification, typing and phylogeny. The 16S rRNA gene sequences, still considered relevant in spite of its proven shortcomings as a basic tool for evaluation of bacterial phylogeny, were used as a baseline for comparative analyses. The statistics of the gene sequences revealed that the variable regions of all three genes have higher resolution capabilities among strains examined compared to the 16S rRNA gene analysis. Phylogenetic analyses based on individual gene sequences and their concatenate enabled to distinguish clusters of species belonging to the genera Acidipropionibacterium, Cutibacterium and Propionibacterium, which corresponds with a recently reported genomic study. Thus, the crucial importance of this study is the economically advantageous classification and typing of propionibacterial isolates and strains through the three gene regions in contrast to the requirement for whole genomic assays.

Gram-negative, rod-shaped bacterial strain ptl-2T, was isolated from soil of the Himalayan valley, India. The bacterial strain ptl-2T has been characterized using a polyphasic taxonomic approach including morphological characterization, fatty acid analysis, biochemical tests, 16S rRNA and nifH gene sequence analysis. 16S rRNA gene sequence analysis showed that the strain ptl-2T belonged to the genus Pseudacidovorax and is closely related to Pseudacidovorax intermedius (99.3 % similarity). It showed <97 % similarity to species of the genera Acidovorax, Alicycliphilus, Xylophilus, Giesbergeria and Simplicispira. The generic assignment has been confirmed on the basis of chemotaxonomic data, which revealed the fatty acid profile, characteristic of the genus Pseudacidovorax, consisting of C16:0 (21.78) and C18:1w7c (19.78) as major fatty acids. Phylogenetic, chemotaxonomic and phenotypic analysis based on signature sequences, DNA-DNA hybridization and physiological characterizations, confirms that strain ptl-2T represents a different species of the genus Pseudacidovorax for which the name Pseudacidovorax austerolens sp. nov. is proposed. The type strain ptl-2T (= CCUG 58759T, = DSM 24877T) has been submitted to two culture collection centres. GenBank accession numbers for the 16S rRNA and nifH sequence of strain Pseudacidovorax austerolens ptl-2T are FJ581042 and GQ249664, respectively.

A thorough polyphasic taxonomic study, integrating genome-based taxonomic approaches, was carried out to characterize the RB5T strain isolated from root nodules of Retama monosperma growing on the coastal dunes of Bousfer Beach (Oran, Algeria). The 16S rRNA gene sequence analysis revealed that strain RB5T had the highest similarity to Pseudomonas granadensis LMG27940T (98.94%) and Pseudomonas gozinkensis IzPS32dT (98.73%). Phylogenetic studies, including both 16S rRNA gene sequence and multilocus sequence analysis using 16S rRNA, gyrB and rpoD housekeeping genes, positioned RB5T in a distinct branch alongside its closest relative, P. granadensis LMG27940T. Phylogenomic analysis using the Bac120 marker set and Type (Strain) Genome Server confirmed the unique position of RB5T and its close relationship with P. granadensis LMG27940T. Similarly, genomic comparisons using average nucleotide identity based on blast (ANIb) and digital DNA-DNA hybridization (dDDH) revealed values of 92.85 and 59.3%, respectively, when compared with its closest relative, P. granadensis LMG27940T. Both values fall below the established species delimitation thresholds of 95-96% for ANIb and 70% for dDDH, providing strong genomic evidence that strain RB5T represents a novel species. Further average nucleotide identity comparisons with unclassified Pseudomonas spp. (384 genomes) and metagenomic-derived genomes from the Genome Taxonomy Database (GTDB) showed values between 84.27 and 89.2%, indicating that strain RB5T belongs to a unique evolutionary line. The genome of RB5T, with a size of 6 311 310 bp and a G+C content of 60%, harbours several key genes associated with plant growth-promoting traits, making it a promising candidate for sustainable agriculture. Phenotypically, RB5T strain is an aerobic, rod-shaped, Gram-negative, non-spore-forming bacterium that is motile with a single polar flagellum. It grows under a wide range of temperature (4-42 °C) and pH (5-10) conditions and tolerates up to 6% (w/v) NaCl. The main cellular fatty acid composition of RB5T includes C16:0, C17:0 cyclo and the summed features 3 consisting of C16:1 ω7c/C16:1 ω6c. Based on the phylogenetic, phenotypic, chemotaxonomic and genome comparison analyses, strain RB5T was identified as a novel species of the genus Pseudomonas, for which the name Pseudomonas retamae sp. nov. is proposed. The type strain is RB5T (=DSM 117471T=LMG 33633T=CIP 112482T).

Forty-one tetrad-forming halophilic lactic acid bacteria were isolated from 7 kinds of fermented foods using MRS agar with 10% NaCl. These bacteria were Gram positive cocci arranged in tetrad form. On the basis of MboI and AluI digested 16S rRNA gene restriction fragment patterns and 16S rRNA gene sequence analysis of the representative isolates, 41 isolates could be divided into two groups (A, B). Twenty-two isolates (group A) were identified as T. halophilus while another nineteen isolates (group B) were identified as T.muriaticus. Histamine formation was determined using HPLC and the partial gene of histidine decarboxylase (hdc) was sequenced. Only one isolate, KS87-14 identified as T. muriaticus, prolifically formed histamine. It indicated that rare strains produced histamine. However, this strain produced histamine 10 times higher than the reported T. muriaticus strains. A structural gene of pyruvoyl dependent histidine decarboxylase (hdcA gene) found mostly in histamine-producing gram-positive bacteria, was detected in KS87-14. Alignment of the partial sequence of hdcA gene from KS87-14 to hdcA gene previously deposited in the database showed that it was similar to the staphylococcal hdcA gene. Seventy-six moderately halophilic bacteria were isolated from 33 samples of fermented fish and salted fish using halobacterium JCM no.377. Only 17 isolates exhibited extracellular ALP activity. Based on their phenotypic characteristics and 16S rRNA gene sequence analyses, these isolates were identified as Staphylococcus saprophyticus (3 isolates), S. napalensis (3 isolates), S. sciuri (1 isolates), Bacillus vietnamensis (1 isolates), B. pumilus (1 isolates), Virgibacillus halodenitrifican (3 isolates), Oceanobacillus iheyensis (1 isolates), Halobacillus mangrovi (1 isolates), H. dabanensis (2 isolates), and the last isolate was closely related to Idiomarina zobellii. They possessed ALP activities ranging from 10.08-81.50 U/ml. The isolate TPS4-2 showed the highest ALP activity. It grew optimally at 30-37oC, pH 8 and in the presence of 10-15% (w/v) NaCl. The predominant ubiquinone was Q-8 and major cellular fatty acids were iso-C15:0 and iso-C17:0. DNA G+C content was 47.0 % mol. Based on 16S rRNA gene sequence analyses, TPS4-2T was closely related to I. zobellii DSM 15924T at 98.7%. It had low levels of DNA-DNA relatedness to the closest type strain I. zobellii DSM 15924T and its unique (GTG)5-PCR genomic fingerprint pattern was different from all closely phylogenic type strains of Idiomarina, therfore it was proposed as I. piscisalsi nov. sp. TPS4-2T was selected for further study due to its novel specie and high ALP production. TPS4-2T could produce both extracellular and intracellular ALP at the beginning of the exponential phase and the highest production was observed at the early stationary phase. TPS4-2T was cultivated in halobacterium JCM377 pH 8.0 at 37C in rotary shaking at 200 rpm. Since intracellular ALP was found higher than extracellular ALP at every sampling hour, therefore, intracellular ALP was further characterized. The maximum intracellular ALP production was at 36 h. The partially purified ALP presented a molecular mass of 53 kDa, calculated by gel filtration. Using p-nitrophenylphosphate as substrate, the Vmax and Km values were 28.01 μmol/min and 0.09 mM, respectively. It had maximal activity in the presence of 0.3 M NaCl, pH 10.0, at 60°C. Stability was fully at pH 7.0-8.0 and 37–55C. The presence of Mg2+ and Ca2+ could stimulate the ALP activity. In addition, the ALP activity was proportional to NaCl concentration in the range of 0-0.3 M. When NaCl concentration was increased 1-4 M, the ALP activity decreased and ceased at 40%. The EGTA could completely inhibit ALP activity. This study indicated that the ALP of TPS4-2T was metalloenzyme that can be activated by Ca2+and Mg2+ ions.